The immune response to entry of a foreign substance into the body consists of secretion by plasma cells of "antibodies" which are immunoglobulin (Ig) molecules with combining sites that recognize particular determinants on the surface of the foreign substance, or antigen, and bind specifically to them. Immunoglobulin is the generic name of various isotypes of antibodies that include IgG, IgM, IgA, IgE, and IgD. The various species of Ig have similarities and differences. For example, all immunoglobulin molecules have a constant portion, i.e., highly conserved (constant) amino acid sequence, within a particular Ig subclass (e.g., IgG.sub.1). This constant region is responsible for various biological effector functions (e.g., complement activation). The portion of the immunoglobulin molecule responsible for immunological specificity (i.e., specific antigen binding) is called the variable region. It is made up of the variable regions of the Ig heavy and light chains. These variable regions differ in amino acid sequence according to the antigenic determinant which the Ig recognizes. Usually, the antibody (Ab) response to an antigen (Ag) is heterogeneous. Upon injection of a body with an immunogen, the body manufactures large numbers of antibodies directed against various determinant sites on the antigen. It is difficult to separate antibodies from conventional antisera containing mixtures of antibodies. It has, therefore, long been a goal to design a continuous source of defined antibodies that recognize and combine with specific antigen determinants.
Hybridoma technology concerns the fusion of myeloma cells with lymphocytes from animals which have been immunized with a particular antigen. The resulting hybridoma cell manufactures monoclonal antibodies that are specific against a single antigenic determinant. Monoclonal antibodies are beginning to replace conventional antisera in standard diagnostic kits for such procedures as the radioimmunoassay. Significant work is also being done to adapt hybridoma technology for therapeutic purposes.
Some properties that flow from an ideal hybridoma cell line are (1) high cloning efficiency; (2) the ability to grow rapidly in a medium supplemented with serum; (3) no secretion of myeloma immunoglobulin (Ig); (4) stable production of large amounts of Ig after fusion; and (5) ability to grow when reinserted into the originating species.
A typical procedure for making hybridomas is as follows: (a) immunize mice with a certain immunogen; (b) remove the spleens from the immunized mice and make a spleen suspension in an appropriate medium; (c) fuse the suspended spleen cells with mouse myeloma cells; (d) dilute and culture the mixture of unfused spleen cells, unfused myeloma cells and fused cells in a selective medium which will not support growth of the unfused myeloma cells or spleen; (e) evaluate the supernatant in each container containing hybridoma for the presence of antibody to the immunogen; and (f) select and clone hybridomas producing the desired antibodies. Once the desired hybridoma has been selected and cloned, the resultant antibody is produced by in vitro culturing of the desired hybridoma in a suitable medium. As an alternative method, the desired hybridoma can be injected directly into mice to yield concentrated amounts of antibody [Kennett, et al., (1981) Ed., Monoclonal Antibodies. Hybridomas: A new dimension in biological analyses, Plenum Press, New York].
Hybridomas produced by fusion of murine spleen cells and murine myeloma cells have been described in the literature by Kohler et al., in Eur. J. Immunol. 6, 511-519 (1976); by Milstein et al. in Nature, 266, 550 (1977); and by Walsh, Nature, 266, 495 (1977).
The technique is also set out in some detail by Herzenberg and Milstein, in Handbook on Experimental Immunology, Ed. Weir (Blackwell Scientific, London), 1979, pages 25.1 to 25.7 as well as in Kennett et al., supra.
Patents relating to monoclonal antibodies against human tumors produced by hybridoma technology include U.S. Pat. Nos. 4,172,124 and 4,196,265. Representative of the art concerning monoclonal antibodies that have specificity for antigens on carcinoma cells are U.S. Pat. No. 4,350,683.
Relative to the parent myeloma cell line employed herein for the fusion event, see Kearney et al, Immunol., 123, 1548-1550 (1978).
DNA mediated transfection experiments using NIH3T3 cells as recipients have led to the identification of human transforming genes from a wide variety of tumor types including established cell lines and primary tumor tissues from cancer patients. To date, approximately 20% of all tumor cells tested have been found to contain transforming genes, termed activated ras genes. The ras genes present in mammalian cells have been demonstrated to be homologous to murine sarcoma viral oncogenes. [Weinberg et al., U.S. Pat. No. 4,535,058; Harvey (1964), Nature, 104: 1104; Kirsten et al. (1967), J.N.C.I., 39: 311]. Thus, genetic sequences homologous to the ras retroviral oncogenes have been found in neoplastic cells as diverse as carcinomas, sarcomas, neuroblastomas and hematopoietic malignancies [reviewed in Cooper, et al. (1983), Biochem. Biophys. Acta. Rev. 738: 9].
Ras genes are found in all nucleated mammalian cells and encode 21,000 molecular weight intracellular membrane proteins designated p21. Viral and cellular ras genes encode membrane bound proteins [Willingham, et al. (1980), Cell, 19: 1005] which bind guanine nucleotides [Scolnick, et al. (1979), PNAS (USA), 76: 5355; Papageorge, et al. (1982), J. Virol., 44: 509; and Finkel, et al. (1984), Cell, 37: 151] and possess intrinsic GTPase activity [McGrath, et al. (1984), Nature, 310: 644; Sweet et al. (1984), Nature 311: 273; Gibbs et al. (1984), PNAS (USA), 81: 5704; and Manne et al. (1985) PNAS, 82: 376]. Transfection experiments using NIH3T3 cells as recipients of human tumor DNA have led to the identification of a family of activated human transforming genes homologous to the ras genes of the Harvey (ras.sup.H) and Kirsten (ras.sup.K) sarcoma viruses. A third member of the ras family designated ras.sup.N has been identified but has not been found to have a retroviral counterpart. Activated ras genes are structurally distinct from their normal homologs, having amino acid substitutions in the protein at positions 12, 13 or 61 [Tabin, et al (1982), Nature, 300: 143; Reddy, et al. (1982), Nature, 300: 149; Bos, et al. (1985), Nature, 315: 716; and Yuasa et al (1983), Nature, 303: 775]. The p21 found in normal cells has the following primary amino acid structure for residues 5 through 16: .sup.5 Lysine-leucine-valine-valine-valine-glycine-alanine-glycine-glycine-valine -glycine-lysine.sup.16. In contrast to normal cells, neoplastic cells have been shown to have amino acid substitutions such as glutamic acid or arginine at position 12 with amino acid residues 5, 6, 7, 8, 9, 10, 11, 13, 14, 15 and 16 being identical to those in normal p21 proteins. [Zarbl et al., Nature (London) 315: 382 (1985); Santos et al., Science, 223: 661 (1984).]
Similarly, neoplastic cells such as the T24 bladder carcinoma have been shown to have an amino acid substitution such as valine at position 12 with amino acid residues 5, 6, 7, 8, 9, 10, 11, 13, 14, 15 and 16 being identical to those found in P21 molecules in untransformed cells.
Previous reports [Furth et al. (1982), J. Virol., 43: 294] have described several rat monoclonal antibodies reactive with normal and activated (mutated) ras p21 proteins in yeast and mammalian cells. One such broadly reactive monoclonal antibody Y13-259 (Id.) has been utilized in Western blot studies to describe elevated levels of ras p21 in human colon and lung carcinoma cells. However, due to the broad cross reactivity of Y13-259, it could not be determined whether the elevated ras in these carcinoma cells was due to activated or normal ras expression [Gallick et al. (1985), PNAS (USA) 82: 1795; Kurzrock et al. (1986), Cancer Res., 46: 1530]. Similarly, monoclonal antibodies raised against ras related synthetic peptides and designated RAP have been shown to be broadly reactive with breast and colon carcinomas; however, this antibody has also been shown to react with normal and mutated ras proteins [Horan et al. (1984), PNAS, 82: 5277; Thor, et al. (1984), Nature, 311: 562].
The subject of this invention is the induction, production and characterization of monoclonal antibodies that react with activated ras proteins containing amino acid mutations at position 12 and that do not react with proteins containing the normal amino acid glycine at position 12. Antibodies E184 and E170 react with activated (oncogenic) ras proteins containing glutamic acid at position 12 instead of glycine, R256 reacts with activated (oncogenic) ras proteins containing arginine at position 12 and DWP reacts with activated (oncogenic) ras proteins containing valine at position 12. Described in this invention are valuable diagnostic tools for the detection staging and classification of primary and metastatic neoplastic cells.